Bottom Line:
Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends.Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length.The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

Affiliation: MRC Centre for Virus Research, University of Glasgow, Glasgow, United Kingdom.

ABSTRACTInfluenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

ppat-1003413-g006: Cell associated long filamentous structures.(A) Tomogram showing a large terminal varicosity and two filamentous particles that appear to contain RNPs at their ends (Insets 1 and 3). An extended helical structure is also seen, possibly M1 (Insets 1 and 2). (B, C) Archetti bodies and the majority of filamentous structures were seen not to have ordered arrangements of RNP at their ends. (D) A filamentous virion containing an extended helical structure that may be M1. See also movie S4.

Mentions:
In addition to Archetti bodies, we saw many filaments that did not terminate with a bulbous varicosity. Some of these filamentous particles had density at their termini reminiscent of the ordered arrangement of genome segments previously described in smaller filamentous particles [21]. The density was not however clearly enough resolved to image the classical ‘seven around one’ arrangement of genome segments in transverse sections (Fig. 6A inset 1 and 3, Movie S4). In these experiments however, we more commonly saw filamentous particles with no distinct density at their termini, rather they had indistinct density along their entire lengths or they were empty (Figs. 6B–C). To evaluate the relative numbers of the various classes of filaments observed we classified 175 long filaments imaged by CET according to the structures seen at their ends. We found that 21.7% of filaments appeared to contain RNPs, 20% of filaments terminated in bulbous Archetti varicosities while 58.3% had no distinct structures at their ends and simply terminated in a hemispherical cap. Thus 78.3% of filaments had no obvious RNP-like structures.

ppat-1003413-g006: Cell associated long filamentous structures.(A) Tomogram showing a large terminal varicosity and two filamentous particles that appear to contain RNPs at their ends (Insets 1 and 3). An extended helical structure is also seen, possibly M1 (Insets 1 and 2). (B, C) Archetti bodies and the majority of filamentous structures were seen not to have ordered arrangements of RNP at their ends. (D) A filamentous virion containing an extended helical structure that may be M1. See also movie S4.

Mentions:
In addition to Archetti bodies, we saw many filaments that did not terminate with a bulbous varicosity. Some of these filamentous particles had density at their termini reminiscent of the ordered arrangement of genome segments previously described in smaller filamentous particles [21]. The density was not however clearly enough resolved to image the classical ‘seven around one’ arrangement of genome segments in transverse sections (Fig. 6A inset 1 and 3, Movie S4). In these experiments however, we more commonly saw filamentous particles with no distinct density at their termini, rather they had indistinct density along their entire lengths or they were empty (Figs. 6B–C). To evaluate the relative numbers of the various classes of filaments observed we classified 175 long filaments imaged by CET according to the structures seen at their ends. We found that 21.7% of filaments appeared to contain RNPs, 20% of filaments terminated in bulbous Archetti varicosities while 58.3% had no distinct structures at their ends and simply terminated in a hemispherical cap. Thus 78.3% of filaments had no obvious RNP-like structures.

Bottom Line:
Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends.Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length.The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

Affiliation:
MRC Centre for Virus Research, University of Glasgow, Glasgow, United Kingdom.

ABSTRACTInfluenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.